Fatty acid uninterrupted by a methylene as anti-inflammatory agents in superficial tissues of mammals

ABSTRACT

The invention concerns fatty acids uninterrupted by a methylene group as anti-inflammatory agents in superficial tissues of mammals, more particularly a pharmaceutical or cosmetic topical composition comprising, as an active ingredient, at least one fatty acid uninterrupted by a methylene group corresponding to formula (I),                    
     an salt or ester thereof, wherein R 1  is a C 1 -C 5  alkyl group and R 2  is a C 2 -C 6  alkyl group. Preferably, the fatty acid uninterrupted by a methylene group is 20:3(5,11,14). The fatty acid uninterrupted by a methylene group is also used for preparing a composition for modulating the metabolism of lipids in the superficial tissue of mammals.

This application is a 371 of PCT/FR98/02584 filed Dec. 1, 1998.

The present invention relates to the new use of a non-methyleneinterrupted fatty acid in the prevention and/or the treatment ofinflammation in superficial mammalian tissues.

BACKGROUND OF THE INVENTION

The use of fatty acids for preventing and/or treating inflammation insuperficial tissues has been described in the literature.

For example, EP 5 472 705 (Prospa B. V.) discloses new pharmaceuticalcompositions for topical use containing esters of ω-3 polyunsaturatedacids in a high concentration and intended for treating psoriasis,phlebitis and related pathologies.

EP 582 239 (Rhône-Poulenc Rorer) discloses topical pharmaceutical andcosmetic compositions containing linoleic acid or derivatives thereof asactive ingredients, and a carrier for transporting the active ingredientinto the skin. These compositions are used for the prophylaxis andtreatment of impure skin, e.g. skin affected by pimples, pustules,urticaria or comedones, of acne and of acne-associated skin disorders.

Furthermore, EP 5 312 834 (Woobang Land Co.) discloses a pharmaceuticalcomposition for treating acne comprising eicosapentaenoic acid and(α-linolenic acid, in a weight ratio of 1:0.1 to 20:0.1 ofeicosapentaenoic acid to α-linolenic acid respectively. These fattyacids may be extracted from natural substances such as fish oil and/orperilla oil, for example.

The term non-methylene-interrupted fatty acid, the acronym for which isNMIFA, refers to a fatty acid with a series of double bonds in which atleast one adjacent pair of double bonds is separated by at least twocarbon atoms, i.e., by a group other than a single methylene group.NMIFAs have been the subject of only a few studies with the aim ofdeveloping an understanding of their incorporation into mammaliantissues and their potential role in the treatment of certain diseases.

For example, JP 61 058 536 (Nippon Oil) discloses a method for purifyingpine nut oil containing at least 10% by weight of5,9,12-cis-octadecatrienoic acid which exhibits a curative effectagainst arterial hypertension. WO 96 05 164 (Broadben Nominees Pty)discloses an anti-inflammatory preparation comprising a purified activefraction, for example 5,11,14,17-eicosatetraenoic acid, isolated from alipid extract of Perna canalicullus or Mytilus edulis.

WO 95 17 987 (The Regents of the University of California) shows thatbroad class of NMIFAs, including 5,11,14-eicosatrienoic acid, may beused in an effective amount for suppressing autoimmune diseases ingeneral, for example rheumatoid arthritis, lupus erythmatosis, multiplesclerosis, myasthenia gravis, and about 30 other diseases currentlyknown.

Incorporation of dietary 5,11,14-eicosatrienoate into various mousephospolipid classes and tissues has been studied. Results show thatfeeding the mice 5,11,14-eicosatrienoate resulted in lower levels of20:4n-6 in the hepatic phosphatidylinositol pool. Because leukotrienesand prostaglandins cannot be formed from 5,11,14-eicosatrienoate due tothe lack of an internal Δ8 double bond, and because 20:4n-6 wasdramatically reduced in some phosphatidylinositol pools, it was expectedthat dietary intake of 5,11,14-eicosatrienate may alter eicosanoidmetabolism, thus reducing potential inflammation in the hepatic system(Biochemica et Biophysica Acta, 1085, 371-376, 1991; J. Nutr. Biochem.,4, 409-420,1993).

So far, the following class of NMIFAs, especially 5,11,14-eicosatrienoicacid has not been reported as being capable of being incorporated intothe lipid fraction of superficial mammalian tissues. Neither has ananti-inflammatory effect in superficial mammalian tissues been expected.

SUMMARY OF THE INVENTION

Accordingly, it has been found that the NMIFAs having the followingformula, wherein the NMIFA is an acid, a salt or an ester, and R¹ is aC₁-C₅ alkyl group and R₂ is a C₂-C₆ alkyl group, may be advantageouslyused for the preparation of a composition intended to modulate themetabolism of lipids in superficial mammalian tissues.

Particularly preferred NMIFAs are those in which R¹ is a C₃ alkyl groupand R² is a C₂-C₆ alkyl group, or in which R² is a C₄ alkyl group and R¹is a C₁-C₅ alkyl group. The most preferred is that in which R¹ is ann-propyl group and R² is an n-butyl group (5,11,14-eicosatrienoic acid,also called 20:3(5,11,14)).

The NMIFAs of the invention may also be used in this context for thepreparation of a composition intended to treat or prevent inflammationsis superficial mammalian tissues by modulating the metabolism of thelipids. The invention also relates to topical pharmaceutical andcosmetic compositions comprising the NMIFAs of the invention as activeingredient. In a last aspect, the invention provides a pharmaceutical,food or cosmetic composition comprising a combination of fish oil andthe NMIFAs of the invention.

The NMIFAs of the invention offer similar advantages as fish oils knownto one skilled in the art. However, they provide the advantage of beingless oxidizable than fish oil, since they have only two methyleneinterrupted bonds as compared to docosahexaenoic acid contained in fishoil having 6 methylene interrupted bonds. In addition the NMIFAs of theinvention are not a substrate for prostaglandin and leukotrieneproduction, whereas prostaglandin and leukotriene can be formed from thefatty acids found in fish oil, such as 20:5n-3 and 22:6n-3. A furtheradvantage over fish oil preparation is the lack of “fishy” odour.

DETAILED DESCRIPTION OF THE INVENTION

“Modulation of the metabolism of lipids” is understood as meaning moreparticularly catabolism of the lipid mediators associated withinflammation, differentiation, proliferation and/or barrier function ofsuperficial tissues.

Furthermore, inflammation of superficial tissues must be understood asthe physiological phenomenon involving the production ofpro-inflammatory cytokines, such as cachectin α (TNFα), by the cells ofsuperficial tissues, for example the keratinocytes and the epithelialcells of the cornea, and the cells of the immune system which arecontained in these tissues (lymphocytes, Langerhans' cells and thelike). The inflammation may result, for example, from an infection, anallergy, a wound and an exposure to radiation and/or irritating agentsand/or sensitizing agents.

The fatty acid which is the subject of this invention is apolyunsaturated fatty acid which is linear and monocarboxylic, with alldouble bonds being cis-double bonds. Several types of nomenclature areused in this specification, and these are as follows.

a) Nomenclature for individual compounds indicating number of carbonatoms and number and position of double bonds, a typical example being“20:4(5,8,11,14)” for arachidonic acid: the number preceding the colonis the total number of carbon atoms, the number immediately followingthe colon is the number of double bonds, and the numbers in parenthesesare the positions of the double bonds, starting from the end of thechain bearing the carboxylic acid group. In all compounds represented inthis manner, except where otherwise indicated, all double bonds are cis.

b) Nomenclature for classes of compounds indicating the location of thedouble bond closest to the methyl end group, a typical example being“n-3” or “n-6”: the number following the dash denotes the position ofthe double bond closest to the methyl end of the molecule, counting fromthe methyl end. Thus, arachidonic acid is in the n-6 class, as is5,11,14-eicosatrienoic acid (20:3(5,11,14,)), whereas5,8,11,14,17-eicosapentaenoic acid (20:5(5,8,11,14,17)) is in the n-3class. This nomenclature is equivalent to “ω” nomenclature in theliterature, “ω” and “n” being interchangeable.

Some of the NMIFAs of the invention are naturally occurring substances.Others may be synthesized according to well known published methodology(see for example Evans et al., Chem. Phys. Lipids, 38, 327-342, 1995).

For example, 20:3(5,11,14) is a naturally occurring substance whichgenerally occurs as one fatty acid in a mixture of fatty acids. ThisNMIFA is found in a wide variety of plants as minor or major fraction ofthe total fatty acid composition. Both the extraction of the mixture offatty acid from their natural sources and the extraction of the20:3(5,11,14) from the resulting fatty acids can be achieved byconventional extraction and purification methods well known among thoseskilled in the art.

The natural sources of fatty acids containing 20:3(5,11,14) areprimarily plant seeds, and prominent among these are conifers andornamental shrubs. The seed oils from these plants are similar to normaledible oils, containing largely oleic, linoleic and linolenic acids, butalso containing useful amounts of NMIFAs. Table 1 lists examples ofseeds whose lipid contents contain significant amounts of 20:3(5,11,14).

TABLE 1 % of % of 20:3 (5,11,14) 20:3 (5,11,14) among total among totalSource fatty acids Source fatty acids Juniperis 14.8 Sciadopitys 15virginiensis verticallata Plarycladus 3 Caltha 23 orientalis palustrisJuniperis 12.3 Calitrus 14 chinesis rhombaidea Torreya 7 Mortierella 7nucifera alpina* Podocarpus 24 Ephedra 22 nagi campylopoda Anemone 10Anemone 6 rivularis leveillei Cimaifuga 6 Erantis 6 racemosa hyemalisGingko 2.2 Pinus 7 biloba silvestris *see the Japanese patent JP5276964(Suntory LTD)

Purification of 20:3(5,11,14) may be in particular achieved by (1)choosing a starting seed source high in total fat content and20:3(5,11,14) content but not containing other contaminating trienes, inparticular α-linolenic acid (18:3n-3) and γ-linolenic acid (18:3n-6)(Podocarpus nagi, Table 1, is such an example); (2) extracting thelipids with isopropanol and chloroform according to the method ofNichols (Biochim. Biophys Acta 70: 417, 1963); (3) conventionaldegumming and decolouring methods; (4) preparing methyl esters with 2%methanolic sulphuric acid according to the method of Christie (p. 52-53,in Lipid Analysis, Pergamon Press, Oxford, 1982); (5) eluting20:3(5,11,14) methyl ester from a silver nitrate impregnated acid-washedFlorisil column with a hexane:ether mixture ranging from 9:1 to 8:2(volume/volume) according to Carroll, J. Am. Oil Chem. Soc. 40: 413,1963; Wilner, Chem. Ind (Lond) October, 30: 1839, 1965; Merck ChromNews4(1): 1995; Anderson, J. Lipid Res. 6: 577, 1965; and Teshima, Bull.Jap. Soc. Scien. Fish. 44: 927, 1978); (6) removing contaminating silverions by the method of Akesson (Eur. J. Biochem. 9:463, 1969); and (7)optionally converting the methyl ester back to the free acid form bysaponification in 1 M potassium hydroxide in 95% ethanol according toChristie (p. 51-52, in Lipid Analysis, Pergamon Press, Oxford, 1982).

In the context of the present invention, the NMIFAs of the invention maybe used in acid, salt or ester form, for example in methyl ester, ethylester, mono-, di-, tri-glyceride or phospholipid ester form. The NMIFAsof the invention may be used in a purified form, or else in the form ofa mixture of fatty acids present in an oil extracted from one of theplants described above, for example.

Administration of the NMIFAs may be achieved by methods known to oneskilled in the art, to any kind of superficial mammalian tissues, thatis to say to cells which make up the skin, the scalp, the eye, or theoral, buccal, nasal and vaginal mucosa, for example.

The NMIFAs may be used for the treatment or prophylaxis of diseases ofthe skin or of the scalp, in particular against inflammations associatedwith, for example, psoriasis, erythema (sunburn), eczema, seborrhoeicdermatitis, alopecia areata, mycosis, acne and other dermatoses.

A composition of the invention, intended for topical applications, maythus comprise an effective amount of the NMIFAs as active ingredient,and a carrier for transporting the active ingredient into superficialmammalian tissues.

The carrier may be any kind of carrier known to one skilled in the artfor cosmetic or pharmaceutical formulations. Liposomes may thus be oneof the carriers used in this context. These molecules include one ormore types of diverse substances including nonpolar lipids, polarlipids, mono- and diglycerides, sulphatides, lysolecithin,phospholipids, saponin, bile acids and salts. Liposomes can exist in theform of emulsions and foams, micelles, insoluble monolayers, liquidcrystals, phospolipid dispersions and lamellar layers. The NMIFAs can beincorporated in the liposome, optionally in conjunction with anappropriate ligand or mimetic agent which binds to specific cellreceptors.

Liposomes are generally formed from standard vesicle forming lipids,which generally include neutral or negatively charged phospholipids anda sterol, such as cholesterol. The selection of lipids is generallyguided by consideration of various factors, such as for example thedesired liposome size and the need for stability of the liposomes in thebloodstream. Typically, the major lipid component in the liposomes isphosphatidylcholine. Partially hydrogenated egg phospatidylcholine is atypical example.

Liposomes typically contain about 5-15 mole per cent of negativelycharged phospholipids, such as phospatidylglycerol, phospatidylserine orphospatidylinositol. Negatively charged phospholipids help preventspontaneous aggregation of the liposomes and thus lower the incidence ofaggregates formed from undersized liposomes. Membrane rigidifyingagents, such as sphingomyelin or a saturated neutral phospholipid, at aconcentration of at least about 50 mole per cent, and 5-15 mole per centof monosialylganglioside, may also by included to provide increasedcirculation of the liposome preparation in the bloodstream.

Liposome suspensions may also include lipid-protective agents to protectthe lipids from free-radical and lipid-peroxidative damage duringstorage. Examples of these agents are lipophilic free-radical scavengerssuch as α-tocopherol, and water-soluble iron-specific chelators such asferrioxyamine.

Liposomes can be prepared by a variety of methods known among thoseskilled in the art. The liposomes can then be sized within a desiredparticle size range and a relatively narrow particle size distribution.A size range which permits the liposome suspension to be sterilized byfiltration through a conventional filter, for example, is about 0.2-0.4microns.

Formulations for administration will generally comprise a solution ofthe compound dissolved or suspended in an acceptable vehicle, theappropriate choices of which will readily occur to those skilled in theart. The formulations may further contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, and wetting agents. The formulations may alsoadvantageously include antioxidants, preferably fat-solubleantioxidants, such as especially tocopherol, tocopherol acetate,butylated hydroxytoluene, butylated hydroxyanisole, ascorbyl palmitateand mixtures thereof.

The cosmetic or pharmaceutical composition may be applied directly tothe superficial tissues such that the NMIFAs diffuse through the cellsand are administered there. This composition can also be injectedunderneath the superficial tissues, for example by means of asubcutaneous injection. In both cases, the application is regarded astopical, that is to say it is applied directly onto or underneath thesuperficial tissues.

Application of the NMIFAs can also be extended to inflammations of theeye, more particularly of the cornea, and also of the mucosa, moreparticularly of the oral, nasal, buccal, anal and vaginal mucosa.Compositions administered orally may affect directly the buccal,oesophageal, gastric and intestinal mucosa, and/or may pass into thebloodstream and be carried directly, for example, to the cells of theskin, the eye or the mucosa.

This composition can also by applied into the nasal passages by means ofa diffuser, a gel and/or a physiological liquid for conventionalflushing of the nasal passages.

It should be pointed out that, depending on the galenical forms usuallyused for topical or oral application, that is to say depending on thedietary, cosmetic and/or pharmaceutical form, the amount of NMIFAssufficient and necessary to observe an anti-inflammatory effect or aneffect as a modulator of the metabolism of lipids may vary considerably.The invention thus relates to the use of the NMIFAs in an amountsufficient for treatment or prevention of inflammations and/or formodulations of the metabolism of lipids of the superficial tissues.

The NMIFAs will thus be administered to a human or an animal, forexample, as a food material to treat oral mucosa. They may thus be usedas replacement for the oils normally used in food formulations orrecipes, or as part of a mixture of oils used in this manner. Thiscomposition can be, for example, a sauce, such as a salad sauce, a tableoil, a mayonnaise, an ice cream, a confectionery composition or afilling paste or spreading paste. The compound can also be administeredas part of a nutritional supplement, such as a tablet or as a capsuletaken orally on a daily basis. Binders, matrices and other conventionaladjuvants normally found in supplements of these types will generally beincluded here as well. Typical dosages for such methods may vary widely,but will most often fall within the range of about 2 mg per kg of bodyweight to about 2000 mg per kg, and more often within the range of about5 to about 500 mg per kg.

The food composition may preferably comprise a carrier to transport theNMIFAs to the large intestine or other region of the gastrointestinaltract. The carrier may be a resistant starch incorporated at a level of2 to 20% by weight. Incorporation can be achieved while drying togetherthe food components, by freeze drying, for example. Incorporation canalso be achieved by microencapsulation. A typical process formicroencapsulation requires the preheating of a low melting point oilabove its melting point (<40° C.), the NMIFAs are then added to themixture, resistant starch is also added together with other ingredientsand binding agents such as gelatin and gums, and the dispersion issprayed into the head of a cooling tower to allow uniform particles toform with an average size typically in the range 20-200 microns. Apreferred form of resistant starch is a high amylose starch, for examplethose disclosed in WO 94/03 049 and WO 94/14 342.

In the field of human and animal cosmetics, the invention also relatesto the use of the NMIFAs for the preparation of a galenical form usuallyused for topical or oral hygiene application, and in particular in theform of oily, alcoholic or aqueous-alcoholic solutions, gels,water-in-oil or oil-in-water emulsions having the appearance of a creamor a gel, if necessary capable of foaming, in the form of an aerosol, oralso in the form of vesicular dispersions comprising ionic and/ornon-ionic lipids. These galenical forms are in all cases prepared by theusual methods in the cosmetic field under consideration. Thiscomposition can be, for example, in particular, a composition forcleansing, protecting, treating or caring for the scalp, for the face,for the neck, for the hands, or for the body (for example in the form ofvanishing creams, night creams, make-up remover creams, suncreams oroils, oils for the body or the face, cleansing milks, make-up removermilks or body milks), a make-up composition (for example a foundation),an artificial tanning composition, a composition for bathing, a shampoocomposition (treatment of the scalp) or a composition for buccal hygiene(mouthwash, toothpaste or chewing gum).

In the field of human or animal pharmacy, in particular dermatology,oto-rhinolaryngology, ophthalmology, gastroenterology and gynaecology,this composition can be, for example, a capsule, a soft gelatin capsule,an emulsion, a pomade, a composition which can be injected underneaththe skin, an ointment, a syrup, a diffuser, an eyewash, a shampoo or amouthwash comprising the NMIFAs for treatment or prophylaxis of theskin, the eyes or the mucosa. The various possible galenical forms arein all cases prepared by the usual methods of the pharmaceutical fieldunder consideration.

The present invention is described in more detail by the examples givenbelow. It goes without saying, however, that these examples are givenonly by way of illustration of the subject matter of the invention, ofwhich they constitute no limitation in any manner. The percentages aregiven by weight, unless indicated otherwise.

EXAMPLE 1

Metabolism of 20:3(5,11,14)

Determination of the profile of 5,11,14 in the human immortalizedkeratinocyte line DK2-NR (described in the patent PCT/EP96/05812) wasachieved as follows.

Confluent cultures of DK2-NR are incubated for 4 days in NR-2 medium(Biofluids Inc. Rockville, Md., USA) with an increasedCa²⁺-concentration (CaCl₂:1.5 mM). Then, the keratinocytes are incubatedtwice in 2 days (medium change with fatty acids after 2 days) in NR-2containing 1 mg/ml bovine serum albumin (BSA, fatty acid free, SigmaInc. St. Louis, USA) and 15 mM of 5,11,14-eicosatrienoic acid methylester (5,11,14-ester) and/or arachidonic acid methyl ester(20;4n-6-ester).

Furthermore, the cells are preincubated for two days with 15 mM20:4n-6-ester (see table No 6-9) and subsequently for 2×2 days with5,11,14-ester and with a mixture of 20:4n-6-ester and 5,11,14-ester.

At the end of the incubation time the cells are washed in HBSS (HanksBalanced Salt Solution) (Gibco, Life Technologies) containing 0.1% BSAand twice in HBSS. The cells are harvested by scraping (cell scraper,Costar Inc.) the cells from the culture dishes. The cells arecentrifuged in 1 ml HBSS (1000 rpm). The cell extraction is carried outin a mixture of hexane and isopropanol (2:1 v/v) containing2,6-di-tert-butyl-p-cresol. The phospholipids are separated bychromatography (silica gel 60) with the eluent chloroform/acetone (96:4v/v). The fatty acid fraction of the phospholipids is then esterified byheating (100° C.) in a solution containing 10% BF₃-methanol (Supelco,Bellefonte, Pa., USA). The methyl esters are separated. The fatty acidsare quantified according to internal standards.

The results listed in table 2 below show that 5,11,14-eicosatrienoicacid is integrated into the total lipids (3.34%).

TABLE 2 Phospholipid fatty acids in cells Treatment with fatty acids5,11, 20:3n-6 20:4n-6 20:5n-3 N° Day 0 Day 2 Day 4 14 % % % % 1 —Ethanol Ethanol 0.00 0.11 0.43 0.04 2 — 5,11,14- 5,11,14- 3.34 0.17 0.470.06 ester ester 3 — 20:4n-6- 20:4n-6- 0.30 0.28 8.25 0.04 ester ester 4— 5,11,14- 5,11,14- 1.04 0.58 9.56 0.00 ester ester 20:4n-6- 20:4n-6-ester ester 5 — 20:4n-6- 20:4n-6- 0.30 0.55 10.39 0.00 ester ester Pre-incuba- tion 6 20:4n- 5,11,14- 5,11,14- 2.12 0.38 5.30 0.03 6-esterester ester 7 20:4n- Ethanol Ethanol 0.00 0.30 5.57 0.01 6-ester 820:4n- 20:4n-6- 20:4n-6- 0.98 1.05 12.90 0.01 6-ester ester ester5,11,14- 5,11,14- ester ester 9 20:4n- 20:4n-6- 20:4n-6- 0.09 0.85 11.800.03 6-ester ester + ester + Ethanol Ethanol

EXAMPLE 2

Effect of 5,11,14-eicosatrienoic Acid on the TNfα-section by UltravioletB Treated Human immortalized keratinocytes

Immortalized human keratinocytes (DK2-NR) are incubated in cultureplates (diameter 3.5 cm) with 1.5 ml NR-2 medium. After reachingconfluency the Ca²⁺-concentration in NR-2 medium is shifted to 1.5 mM.After 4 days, the cells are incubated 2×2 days in NR-2 withouthydrocortisone, with a high concentration of Ca²⁺ (1.5 mM) and variousfatty acids (i.e. 18:2n-6, 18:3n-3, 20:3(5,11,14)). At the end of theincubation period the NR-2 medium is removed and stored at 37° C. tore-feed the cells after the UVB-treatment (conditioned NR-2 medium). Thecell cultures are washed twice with HBSS. For the UVB-treatment thecells are incubated in 0.5 ml HBSS. The cell cultures are treated with1.5 kJ UVB (Philips, TL100, maximum emission at 313 nm). Then, HBSS isreplaced with the corresponding conditioned NR-2 medium. The supernatantis collected after 24 hours and is stored at −20° C. The secreted TNFαconcentration in the supernatant is quantified by ELISA (PelikineCompact, CLB Amsterdam, The Netherlands).

As listed in table 3 it could be shown that 5,11,14-eicosatrienoic acidinhibits the secretion of TNFα (9 pg/ml) in UVB-treated humanimmortalized keratinocytes compared to controls (11.6 pg/ml).

TABLE 3 Secreted TNFα [pg/ml] ± SD No UVB + control 0.0 0 (EtOH) UVB +control (EtOH) 11.6 0.009 UVB + 5,11,14 9.0 0.005 UVB + 18:3n-3 14.50.015 (α-linolenic acid) UVB + 18:1n-9 12.1 0.005 (oleic acid) UVB +hydrocortisone 2.0 0.004 (0.5 mg/ml)

EXAMPLE 3

Anti-inflammatory Effect of Other NMIFAs

Determination of the anti-inflammatory effect of 18:3(5,11,14),18:3(3,9,12), 19:3(5,11,14), 19:3(4,10,13), 21:3(5,11,14),21:3(6,12,15), 22:3(5,11,14) and 22:3(7,13,16) in the human immortalizedkeratinocyte line DK2-NR (described in the patent PCT/EP96/05812) wasachieved as described in Example 2 above. The results show thatanti-inflammatory effects are exhibited.

EXAMPLE 4

Anti-inflammatory Activity of Podocarpus nagi Methyl Ester FollowingTopical Administration to the Mouse

The anti-inflammatory activity of Podocarpus nagi methyl ester(containing 26% of C20:3 5,11,14; and containing 0.5% of α-tocopheroland 0.2% of ascorbyl palmitate) was evaluated in the arachidonicacid-induced aural oedema test in the mouse.

Podocarpus nagi methyl ester corresponds to the oil extracted from seedsof Podocarpus nagi, the extraction being carried out in accordance withthe method described in the two paragraphs following Table 1 of thedescription.

The exact protocol is as follows: Podocarpus nagi methyl ester or thePodocarpus nagi control oil is administered topically to the right earof mice at a concentration of 20% (4 micrograms in 20 microliters ofacetone) once a day for 5 days. Each group contains 5 mice and one groupreceives only acetone. Arachidonic acid at a concentration of 2% inacetone is administered 1 hour after the final application of thetreatments, and the thickness of the ear is measured with the aid of amicrometer (oditest) 1 hour after the administration of the arachidonicacid.

The Podocarpus nagi control oil corresponds to Podocarpus nagi methylester as described above in which the C20:3 5,11,14 has been essentiallyreplaced by oleic acid (C18: 1n-9).

Results

The aural oedema corresponds to the increase in the thickness of the earin relation to the group having received acetone only.

Student test Product Aural oedema Inhibition of (versus administered (mm10⁻²) oedema (%) arachidonic topically average ± mse average ± mse acidgroup) Arachidonic 11.75 ± 1.75 — — acid (AA) 2% Indomethacin 5%  1.00 ±0.48 91.49 P < 0.001 + AA Podocarpus nagi  5.00 ± 0.95 57.45 P < 0.01methyl ester + AA Podocarpus nagi 12.00 ± 2.10 −2.13 P > 0.05 (not oil +AA significant) mse: mean standard error

Indomethacin, a non-steroidal anti-inflammatory used as a positivecontrol in the test at a concentration of 5% in acetone, greatlyinhibits the oedema induced by arachidonic acid. Podocarpus nagi methylester at a concentration of 20% provides 57% inhibition of thearachidonic acid-induced oedema. These results show that Podocarpus nagimethyl ester exhibits an anti-inflammatory activity when it isadministered topically to the skin of mice.

EXAMPLE 5

Analysis of the Lipids in the Skin Following Topical Administration ofPodocarpus nagi Ethyl Ester to the Mouse

Podocarpus nagi ethyl ester corresponds to the oil extracted from seedsof Podocarpus nagi, the extraction being carried out in accordance withthe method described in the two paragraphs following Table 1 of thedescription.

The exact protocol is as follows: Podocarpus nagi ethyl ester (withantioxidants) or the control mixture (with the same composition asPodocarpus nagi ethyl ester but in which the C20: 3 5,11,14 isessentially replaced by C18: 1n-9) is administered topically to theright ear of mice at a concentration of 20% (4 micrograms in 20microliters of acetone) once a day for 5 days. Each group contains 4mice. On the left ear, the mice receive only acetone. 2 hours after thefinal administration, the mice are sacrificed and biopsies are carriedout on the ears thus treated for analysis of the cutaneous lipids.

The incorporation of C20: 3 5,11,14 into the phospholipids of the skinof the ear of mice was analysed as follows:

Extraction of the lipids: 20 mg of each biopsy is extracted with 0.8 mlof H₂O, 1 ml of CHCl₃ and 2 ml of MeOH. Following centrifugation (3minutes at 1000 g) the residue is re-extracted with 1 ml of CHCl₃, thesupernatant is filtered through a glass frit and washed with 1 ml of0.88% KCl. The organic phase is extracted and concentrated undernitrogen and redissolved in 2 ml of CHCl₃.

Separation of the phospholipids (PL) and the neutral lipids (NL): thedifferent classes of lipids were separated by solid-phase chromatographyon 3 ml columns containing 500 mg of silica (Supelco 57010). The samplesas described above (in 2 ml of CHCl₃) were introduced into these washedcolumns. The NL were obtained with 2 ml of CHCl₃ and the PL with 4 ml ofCHCl₃/MeOH (2/1) and 4 ml of MeOH. The samples are concentrated undernitrogen and redissolved in 10 ml of CHCl₃.

Thin-layer chromatography of the phospholipids: the samples of PLobtained above are introduced onto 10×10 thin-layer chromatographyplates (Merck 1.13727) and the PL are separated in CHCl₃/MeOH/aceticacid/H₂O (50/37.5/3.5/2.9). The PL are identified using referencesamples.

Methylation of the phospholipids: the samples are subsequentlymethylated with 200 μl of methanolic HCl/hexane (4/1) containing 0.4 μgof C17:0 for 16 hours at 65° C. The reaction is subsequently neutralizedwith 0.5 ml of 6% K2CO3. The methyl esters (ME) are extracted twice with300 μl of hexane, concentrated under nitrogen and redissolved in 5 μl ofisooctane for analysis by gas chromatography.

Gas chromatography of the phospholipids: the ME are injected into theinstrument Hewlett Packard GC Model 6890, equipped with a samplecollector and a capillary column.

RESULTS: Fatty acids in the lipid pools after topical application of theethyl esters of controls* (control) and ethyl esters of Podocarpus nagi(seed) on the mouse ear.

Composition of the ethyl esters admini- stered topically PhospholipidsPercentages Control Seed Seed Fatty acids (+**) (+**) Control (+**)c16:0 5.4 2.4 14.6 8.2 c16:1n-7 0.3 1.6 5.9 c18:0 1.9 1.4 14.8 7.6c18:1n-9 43.9 16.4 20.0 15.5 c18:2n-6 48.5 43.1 16.8 27.0 c18:3n-3 0.00.3 c20:2 5, 11 0.4 0.0 0.2 c20:3 5,11,14 26.3 0.2 13.6 c20:2n-6 8.6 0.84.4 c20:3n-6 0.7 0.2 c20:4n-6 9.9 2.2 c20:5n-3 0.1 0.3 0.0 c23:0 0.3 0.0c22:2n-6 0.2 0.0 c22:4n-6 1.1 0.0 c22:5n-3 1.0 0.0 c22:6n-3 9.3 1.0Σ(c12,c13,c14, 1.9 2.5 c15:0) Σ(c20, c22, c24, 4.4 9.7 c26:0)Σ(c20,c22,c24:1) 1.5 2.1 1.7 Total 100.0 100.0 100.0 100.0*Saffron/sunflower/apricot (43/7/50) **Antioxidants: 0.5% by weightα-tocopherol and 0.2% by weight ascorbyl palmitate

The C20:3 5,11,14 increases in the phospho-lipids whereas the 20:4n-6(arachidonic acid) decreases. These results show that C20:3 5,11,14 isincorporated into the skin phospholipids following topical applicationto the mouse.

The replacement of 20:4n-6 by C20:3 5,11,14 seems to correlate to theanti-inflammatory properties of C20:3 5,11,14 evidenced after topicalapplication in the model of arachidonic acid-induced aural oedema in themouse (Example 4).

Example 6

Body Milk (Oil-in-water Emulsion)

Oily Phase:

Glyceryl stearate/PEG-100 stearate 1% (Arlacel 165 sold by ICI)(emulsifier) Polysorbate 60 (emulsifier) 0.8% Hydrogenated polyisobutene2% Stearic acid 1% Oil from seed of Ephedra campylopoda 8%

Aqueous Phase:

Glycerin 3% Carbomer (carbopol 941 sold by 0.3% Goodrich) (thickener)Triethanolamine (neutralizing agent) 0.3% Preservative 0.3% Water up to100%

The emulsion is prepared by incorporating the oily phase in the aqueousphase while stirring. The body milk provides good protection of the skinagainst inflammations.

EXAMPLE 7

Care Fluid (Oil-in-water Emulsion)

Oily Phase:

Methylglucose sesquistearate (emulsifier)  2% Cyclomethicone 13% Oilfrom seed of Podocarpus nagi  2% Perfume  0.2% PEG 20 methylglucosesesquistearate (emulsifier)  2%

Aqueous Phase:

Xanthan gum (thickener) 0.2% Polyacrylamide/isoparaffinC₁₃-C₁₄/laureth-7 0.8% (Sepigel 305 sold by Seppic) (thickener)Preservative 0.3% Water up to 100%

The emulsion is prepared as describe in Example 6. A white fluid isobtained which provides good protection of the skin againstinflammations.

Example 8

Care Cream (Water-in-oil Emulsion)

Oily Phase: (A)

Polyglyceryl-4 Isostearate/cetyl dimethicone  4% Copolyol/hexyl laurate(Abil WE 09 sold by Goldschmidt) (emulsifier) Isohexadecane  5% Oil fromseeds of Caltha palustris 10% Cyclomethicone  3.5%n-octanpyl-5-salicylic acid  1% Perfume  0.15%

Aqueous Phase: (B)

Glycerin 10% Cellulose gum  0.5% Magnesium sulphate  0.65% Preservative 0.3% Water up to 100%

For preparing the emulsion, constituents of phase A are heated at 80° C.until completely dissolved, and are refrigerated at 65° C. Phase B isheated at 65° C., phase A is poured therein while stirring, then themixture is refrigerated. A white fluid is obtained which provides goodprotection of the skin against inflammations.

What is claimed is:
 1. A topical pharmaceutical or cosmetic compositioncomprising, as an active ingredient, at least one substance selectedfrom the group consisting of non-methylene-interrupted fatty acidshaving formula (I), salts and esters thereof, along with a carrier fortransporting the active ingredient into a superficial mammalian tissue,

wherein R¹ is a C₁-C₅ alkyl group and R² is a C₂-C₆ alkyl group.
 2. Atopical pharmaceutical or cosmetic composition according to claim 1,wherein R¹ is a C₃ alkyl group and R² is a C₂-C₆ alkyl group, or inwhich R² is a C₄ alkyl group and R¹ is a C₁-C₅ alkyl group.
 3. A topicalpharmaceutical or cosmetic composition according to claim 2, wherein thenon-methylene-interrupted fatty acids are 20:3(5,11,14).
 4. A topicalpharmaceutical or cosmetic composition according to claim 1, wherein thenon-methylene-interrupted fatty acids have been previously purified,synthesized, or are present in a mixture of fatty acids.
 5. Apharmaceutical, food or cosmetic composition, comprising a combinationof a fish oil and at least one substance selected from the groupconsisting of non-methylene-interrupted fatty acids of formula (I),

salts and esters thereof, wherein R₁ is a C₁-C₅ alkyl group and R₂ is aC₂-C₆ alkyl group.
 6. A composition according to claim 1, furthercomprising liposoluble anti-oxidants.
 7. A method to modulate themetabolism of lipids in superficial mammalian tissues comprisingadministering an effective amount of at least one substance selectedfrom the group consisting of non-methylene-interrupted fatty acids offormula (I), salts and esters thereof to a mammal in need of suchmodulation,

wherein R¹ is a C₁-C₅ alkyl group and R² is a C₂-C₆ alkyl group.
 8. Amethod to treat or prevent inflammation in superficial mammalian tissuescomprising administering an effective amount of at least one substanceselected from the group consisting of non-methylene-interrupted fattyacids of formula (I), salts and esters thereof to a mammal in need ofsuch treatment of prevention,

wherein R¹ is a C₁-C₅ alkyl group and R² is a C₂-C₆ alkyl group.
 9. Amethod according to claim 7, wherein the non-methylene-interrupted fattyacids are 20:3(5,11,14).
 10. A method according to claim 8, wherein thenon-methylene-interrupted fatty acids are 20:3(5,11,14).
 11. A topicalpharmaceutical or cosmetic composition according to claim 1, wherein thecarrier is a liposome.
 12. A topical pharmaceutical or cosmeticcomposition according to claim 1, further comprising a pharmaceuticallyor cosmetically acceptable vehicle.
 13. The method of claim 7, whereinthe at least one substance is administered along with a carrier fortransporting the active ingredient into a superficial mammalian tissue.14. The method of claim 8, wherein the at least one substance isadministered along with a carrier for transporting the active ingredientinto a superficial mammalian tissue.
 15. The method of claim 13, whereinthe carrier is a liposome.
 16. The method of claim 14, wherein thecarrier is a liposome.
 17. The method of claim 7, wherein the at leastone substance is administered along with a fish oil.
 18. The method ofclaim 8, wherein the at least one substance is administered along with afish oil.
 19. The method of claim 13, wherein the carrier is a resistantstarch.
 20. The method of claim 14, wherein the carrier is a resistantstarch.
 21. A topical pharmaceutical or cosmetic composition accordingto claim 1, wherein the carrier is a resistant starch.
 22. A topicalpharmaceutical or cosmetic composition according to claim 1, furthercomprising a fish oil.